现代食品科技2017，Vol.33Issue(8)：36-40,5.DOI:10.13982/j.mfst.1673-9078.2017.8.007
鉴定活性维生素D细胞模型的构建
Construction of a Cell Model for the Identification of Active Vitamin D
摘要
Abstract
A eukaryotic expression vector containing a vitamin D receptor (VDR) fusion protein was constructed,enabling the creation of a cell system that could stably express VDR fusion protein,and a mature cell model for the identification of food and drugs containing active vitamin D was established.Specific primers were designed and synthesized to amplify the VDR gene,the gene amplification products were cloned into a pcDNA 3.1/His eukaryotic expression vector,and the vector was then transfected into HEK293 cells.Immunoprecipitation (IP)was used to identify the expression efficiency of the fusion protein.Using the successfully constructed pcDNA 3.1/VDR-His vector,the transfected HEK293 cells were treated with an active form of vitamin D,1,25-dihydroxyvitamin D3,and then total protein and mRNA were quantified.Western blot (WB) analysis and quantitative real-time PCR (qRT-PCR) were used to detect the expression of the VDR fusion protein and its target gene,CYP24A1,respectively.The IP results showed that the VDR fusion protein had transcription activity.WB and qRT-PCR results showed that 1 nmol/L 1,25-dihydroxyvitamin D3 could effectively activate VDR fusion protein expression in the transfected cell system and significantly increase CYP24A1 mRNA expression (p ＜ 0.01).This cell model was used to determine the activity of vitamin D drugs,and the results suggested that the vitamin D activity of different drugs was related to drug type.The pcDNA 3.1/VDR-His fusion-protein expression vector system was successfully constructed,and a cell model to identify active vitamin D in food and drugs was successfully established using the working vector.关键词
维生素D受体/融合蛋白/细胞模型Key words
vitamin D receptor/fusion protein/cell model引用本文复制引用
程佳,王永吉..鉴定活性维生素D细胞模型的构建[J].现代食品科技,2017,33(8):36-40,5.基金项目
陕西省教育厅专项科研计划项目(12JS029) （12JS029）
陕西省科技创新团队项目(2012KCT-29) （2012KCT-29）
